Type I diabetes is a systemic autoimmune disease. Transplantation of the hematopoietic stem cell (HSC) from disease-resistant donors reverses the autoimmunity in non-obese diabetic (NOD) mice before they develop insulin-dependence. The focus of this proposal is to identify strategies to allow the clinical application of HSC chimerism to treat diabetes. The morbidity and mortality associated with conventional bone marrow (BM) transplantation could not be accepted in the clinical application of HSC chimerism to reverse diabetes. Conditioning of the host to allow engraftment of the HSC requires a combination of cytoreductive and immunosuppressive agents. Strategies to achieve HSC chimerism with partial conditioning would be essential if mixed HSC chimerism is to be applied clinically to treat type I diabetes. A precise definition of which cells must be removed from the host microenvironment will allow specific targeting of these cells to make space to achieve HSC chimerism. In Aim I we will establish a minimal conditioning approach to mixed HSC chimerism in NOD mice. The composition of the donor marrow inoculum also influences engraftment. In normal mice, when BM is T-cell depleted (TCD), more conditioning and a higher cell dose are required for engraftment of allogeneic HSC. In NOD mice, TCD BM does not engraft. In Aim II we will identify which cell types in donor BM (T-cells vs. facilitating cells) are critical to engraftment of allogeneic HSC in NOD recipients. By optimizing conditioning of the autoimmune recipient and the composition of the donor BM, we will achieve engraftment with minimal recipient morbidity. NOD mice exhibit a number of hematopoietic abnormalities that result in a relative alloresistance to engraftment: (1) HSC from NOD mice do not produce functionally mature antigen presenting cells (APC), (2) APC from NOD mice do not delete potentially autoreactive T-cells during their development, (3) NOD mice require a higher cell dose and more conditioning for engraftment, (4) NOD mice fail to engraft after TCD of the donor marrow, and (5) once engraftment occurs in NOD recipients, the level of donor engraftment approaches greater than 95 percent, suggesting that disease-resistant BM has a competitive advantage over NOD BM. In Aim III we will characterize the hematopoietic defects in NOD mice, focusing on the mechanisms responsible and strategies to overcome the defects. The overall focus of this proposal is to identify factors which will allow the clinical application of mixed HSC to halt the autoimmune process in diabetes before the onset of terminal complications.